The three studied species (F. kalenesos, C. styx and Z. yong) are representatives of three aberrant kinorhynch genera (Fig. 1). Aberrant kinorhynchs show distinct morphological characters that differ from the typical kinorhynch morphology such as: modified introverts with fewer spinoscalids and very elongated tentacle-like primary spinoscalids; weakly developed or, in some cases, absent neck; elongated trunk with thin, flexible cuticle and less distinct segmentation. For additional information on aberrant kinorhynch species see Herranz et al. [27]. For details on the phylogenetic position of the selected aberrant kinorhynchs see Sørensen et al. [40]. Comprehensive descriptions of cuticular morphology of F. kalenesos can be found in Dal Zotto et al. [42] and Rucci et al. [43]; of C. styx in Higgins [44], Neuhaus and Kegel [45] and Herranz et al. [41], of Z. yong in Altenburger et al. [46].
Due to the numerous similarities found in the nervous system architecture in the three studied species, a detailed neuroanatomical description is only provided for F. kalenesos while differences to this are given for C. styx and Z. yong. Abbreviations: i, introvert; t, trunk.
Neuroanatomy of F. kalenesos
Acetylated α-tubulin-like immunoreactivity
Acetylated α-tubulin-like immunoreactivity (α-tub-LIR) was studied in twenty specimens of F. kalenesos. α -tub-LIR is consistent in all studied specimens showing a circumpharyngeal brain neuropil (np), of ca. 15 μm width that narrows on the ventral side, composed of multiple densely arranged transverse neurites (Fig. 2A–D). The position of the brain varies depending on the level of introvert retraction, however when the introvert is everted the brain is located inside the neck or the first trunk segments.
From the anterior part of the brain neuropil ten longitudinal bundles (lnb) arise radially, extend anteriorly and bend 180° towards the body wall before extending posteriorly along the trunk (Fig. 2A–D). Of the ten neurite bundles the two ventromedial ones are the most prominent; they fuse in segment 1 forming the ventral nerve cord (vnc) (Fig. 2B, C). The remaining eight neurite bundles also fuse two by two at the level of segments 1-2, forming two ventrolateral and two subdorsal nerves (Fig. 2A, B). The ventral nerve cord extends in a midventral position along the trunk reaching the posterior end of segment 9, where it splits into four neurite bundles (vncn), two to the left and two to the right (Fig. 2B, C, E, F, H). Two of the ventral nerve cord neurite bundles extend laterally towards segment 10 and two towards segment 11 encircling each segment and connecting to the subdorsal nerves (Fig. 2A, B, G, H). The two ventrolateral nerves (vln) extend from segment 2 to segment 10 and the two subdorsal nerves (sdn) from segment 2 to segment 11 (Fig. 2A, B, G, H). All longitudinal cords are connected through a bundle of transverse ring neurites (tn). These neurite bundles originate from left and right sides of the ventral nerve cord and encircle segments 1-9 and the neck (Fig. 2A, B, E, F); the α-tub-LIR in the neck was very weak though. The transverse neurite bundles seem to split from ventrolateral to sublateral positions of most segments, yet the pattern is not consistent in every segment (Fig. 2J). Some segments also show additional thin transverse neurites in ventromedial positions (Fig. 2F).
Out of the five nerve cords the ventral nerve cord is the only one associated with paired clusters of nuclei. Nuclei are situated along the ventral nerve cord in the anterior half of each segment, being more numerous and densely clustered anterior to the arising point of the transverse neurite bundles (Fig. 2E, F). These paired clusters are interpreted as ganglia, present in segments 1-9; however, in segment 1 the nuclei form a unique dense cluster that seems to extend along the entire segment length. Whether this is a single large ganglion or two fused ganglia needs to be determined with ultrastructural studies. The intersegmental region of the ventral nerve cord does otherwise not have nuclei associated (Fig. 2E).
Most cuticular structures such as introvert spinoscalids, mouth cone styles, trunk spines and sensory spots show intrinsic α-tub-LIR. Every introvert spinoscalid has a neurite or neurite bundle arising from the tip and connecting to the anterior part of the neuropil, except for the primary spinoscalids (psp) which show two prominent neurite bundles each (psn) (Figs. 2D, 3E, F). Inner and outer mouth cone oral styles, show neurites (isn, osn respectively) that arise in the tip of each style and bent 180° to connect to the posterior end of the neuropil (Fig. 2D). Inner and outer oral style neurites are encircled by a circular neurite bundle (mcnr) situated at the base of the inner oral styles. All trunk acicular spines (lateroventral and middorsal) show neurites (sn) originating in the tip of each spine, extending along its length and connecting to the transverse neurite of the corresponding segment (Fig. 2I). Lateral terminal spines and lateral terminal accessory spines of segment 11 also have neurites (tsn) that extend from the tip of each spine and connect to the ventral nerve cord neurites of this segment (Fig. 2A, B). Each sensory spot of the trunk shows two short parallel neurites (ssn), one of them extending from the external pore towards the transverse neurite bundle of the same segment (Fig. 2F–H).
Two pairs of α-tub-LIR ring-like structures (marked with arrowheads in Fig. 2C, D) were found in the dorsal part of the introvert, anterior to the brain neuropil, and ventromedially at the level of the brain neuropil (Fig. 2C, D). Due to the position of the rings, which overlap with the multiple neurite bundles emerging from the spinoscalids, it is not possible to see if there is any connection from the rings to the brain neuropil or the introvert. Specimens co-labelled with DAPI show that each of the ring-like α-tub-LIR structures surrounds a single cell nucleus. The two dorsal cells are regarded as part of the anterior brain region and the two ventral cells seem to be either part of the anterior brain region or the neuropil (Fig. 3E, F).
Non-neuro-specific α-tub-LIR was detected laterodorsally on segments 8-9 congruent with the position of the two nephridia.
Serotonin-like immunoreactivity
Serotonin-like immunoreactivity (5HT-LIR) was studied in sixteen specimens of F. kalenesos. All specimens show a similar 5HT-LIR present in the brain neuropil, at the base of the mouth cone and in the ventral nerve cord (Fig. 3). Within the neuropil, at least four to five serotonin immunoreactive rings can be detected; of these, the first three rings are ventrally incomplete (inr). The first ring extends from a pair of big ventromedial somata (vms). Associated somata of the second and third rings are not identified, but their two pairs of neurites extend anteriorly before bending 180 degrees and continuing posteriorly, first via the ventral convergent neurites (cne) and then via the fused ventral cord (vnc), until segment 10 (Fig. 3A–F). Remaining neuropil rings are complete (cnr) and are located posterior to the incomplete rings (Fig. 3A–C). Additionally, 5HT-LIR neurites are present forming a ring at the base of the mouth cone within the mouth cone nerve ring (mcnr) (Fig. 3A, C, D). Serotonin immunoreactive somata are associated with the anterior and posterior brain regions, as well as the ventral nerve cord. Within the anterior brain region, besides the ventromedial somata, there are two additional pairs of somata in laterodorsal and middorsal positions (asb) that connect with the neuropil (Fig. 3A–E). Within the posterior brain region there is one pair of laterodorsal somata (psb) connected to the neuropil (Fig. 3A–F). Along the ventral cord, paired somata (vncs) are present in the ganglia of segments 3 and 6, and unpaired median somata in the ganglia of segments 5 and 8 (Fig. 3A–C, G).
FMRFamide-like immunoreactivity
FMRFamide-like-immunoreactivity (FMRF-LIR) was studied in four specimens of F. kalenesos. FMRF-LIR is consistently found in the brain neuropil, associated somata, ventral nerve cord and subdorsal nerves (Fig. 4). Weaker FMRF-LIR is present in the intersection areas between the ventrolateral nerves and transverse neurites of each segment, and in the transverse neurites of segment 9 (Fig. 4). Within the brain FMRF-LIR is localized in the neuropil (Fig. 4A–D, F). Multiple pairs of FMRF-LIR somata (ca. 10-12) similar in size are radially distributed in the anterior brain region (asb) (Fig. 4A–D, E). These neurons are interpreted as bipolar, with their somata seemingly projecting neurites into the anteriormost part of the neuropil as well as long neurites towards the introvert scalids. Additionally, five pairs of FMRF-LIR somata (two midventral, two midlateral and one subdorsal pair) are present in the posterior brain region connecting with the posteriormost part of the neuropil (Fig. 4A–D, G). The two convergent neurite bundles (cne) that emerge from the brain and the ventral nerve cord show several neurites with FMRF-LIR. The ventral nerve cord was labelled along its length until segment 10. Paired FMRF-LIR somata associated with the ganglia of the ventral nerve cord are present in segments 3-5, 7, 8 and 9 (Fig. 4A, B, H, I). All the somata are similar in size except for the somata of segment 3 which are larger (Fig. 4B, H). Moreover, at least three to four pairs of FMRF-LIR somata were found in subdorsal position on segment 10 associated with the transverse neurite bundle (Fig. 4 A, J).
Neuroanatomy of C. styx
Acetylated α-tubulin-like immunoreactivity
Acetylated α-tub-LIR was studied in eighteen specimens of C. styx. In general, the α-tub-LIR pattern in C. styx agrees with the description provided for F. kalenesos and is therefore not included herein (Fig. 5), instead, only the diverging neuroanatomy is addressed.
The position of the brain in C. styx varies depending on the level of introvert retraction, reaching segment 5 when fully retracted. However, all the studied specimens with the introvert everted had the brain also located inside the trunk, in positions varying from segment 1-3 (Fig. 5D–F). The ventral nerve cord ganglia are paired, elongated and extend evenly along the segment length in parallel to the ventral nerve cord leaving short somata-free areas between segments (Fig. 5C–C’).
C. styx lacks a neck and therefore the cuticle of the anterior part of segment 1 continues directly into the soft cuticle of the introvert (Fig. 5A, B). This is different compared with F. kalenesos, which has a distinct neck region with a transverse neurite bundle encircling it.
Besides the lateral terminal and lateral terminal accessory spines, C. styx has a long midterminal spine (mts) in segment 11 that shows α-tub-LIR along its length. From the tip of the spine a long neurite extends towards the transverse neurite bundle of segment 11 (Fig. 5A).
C. styx has two types of sensory spots in the trunk (type 1 and type 5) with different α-tub-LIR patterns. Type 1 sensory spots show two neurites that connect to the transverse neurite bundle of the corresponding segment (ssn1); instead, sensory spots type 5 only have a single neurite (ssn5) (Fig. 5G–H’). Additionally, C. styx has a dorsal organ located in a middorsal position in between segments 5 and 6 that shows α-tub-LIR only in the proximal part (don) (Fig. 5A). Among all kinorhynchs, this organ is unique for C. styx. Details of the dorsal organ morphology are described and illustrated in Herranz et al. [9] and thus not included in this study.
Serotonin-like immunoreactivity
Serotonin-like immunoreactivity was studied in twelve specimens of C. styx. All specimens show a common 5HT-LIR pattern only detected in the brain neuropil and the ventral nerve cord (Fig. 6A). In the brain neuropil there are ca. six immunoreactive neurites forming a ring, where the three first neurites are incomplete ventrally (Fig. 6A, C, D). The anteriormost incomplete ring is composed of neurites that extend from a pair of ventromedial somata (vms). The neurites of the two following posterior rings extend anteriorly towards the introvert and bend 180° to join the convergent neurite bundles (cne) that form the ventral nerve cord. Besides the ventromedial somata, there is an additional pair of 5HT-LIR somata within the anterior part of the brain located in laterodorsal position that projects neurites into the anterior part of the neuropil (Fig. 6A, C, D). No immunoreactive somata were found in the posterior brain region. Only one pair of 5HT-LIR somata was found associated with the ventral nerve cord (vncs) in segment 5 (Fig. 6A, B).
FMRFamide-like immunoreactivity
FMRF-LIR was studied in six specimens of C. styx. Stainings of the posteriormost trunk segments [7,8,9,10,11] were not successful. FMRF-LIR is consistent in the brain neuropil, associated somata, base of the mouth cone and the ventral nerve cord (Fig. 7). The brain neuropil shows multiple FMRF-LIR neurites along its full width (Fig. 7A–C). Associated with the neuropil, multiple sets of somata are located in the anterior and posterior brain regions but fewer than in F. kalenesos. The anterior brain region shows at least six pairs of bipolar somata (asb), radially arranged and similar in size, that project neurites into the anterior part of the neuropil as well as anteriorly towards the introvert (Fig. 7A–E). The posterior brain region only shows two pairs of ventromedial somata (psb) which connect with the posterior part of the neuropil (Fig. 7A–C). FMRF-LIR was also detected as a ring at the base of the mouth cone, within the mouth cone nerve ring (mcnr). The ventral nerve cord shows FMRF-LIR at least from segments 1-7. FMRF-LIR pairs of somata are only situated at the posteriormost region of segments 3-4 (Fig. 7A, F), and seem to be part of the ventral nerve cord ganglia.
Neuroanatomy of Z. yong
Acetylated α-tubulin-like immunoreactivity
Acetylated α-tub-LIR was studied in twelve specimens of Z. yong, all of them showing identical patterns (Fig. 8). The pattern of α-tub-LIR in Z. yong is very similar to F. kalenesos and C. styx, and therefore this description will only focus on the differential neuroanatomy.
The position of the brain in Z. yong varies depending on the degree of eversion/retraction of the introvert. In F. kalenesos and C. styx the brain can remain inside the anterior trunk segments or the neck even when the introvert is everted. All specimens of Z. yong had the introvert withdrawn (Fig. 8D) and thus the position of the brain relative to the everted introvert could not be examined (Fig. 8A, B shows introvert and mouth cone extended to ease comparisons among species, the position of the brain herein should not be considered).
The ventral nerve cord in Z. yong extends from segment 1-10. Clusters of nuclei are distinct in most segments forming paired ganglia (dashed areas in Fig. 8C, E), except in segments 1 and 10 (Fig. 8C). Unlike F. kalenesos and C. styx, most of the ventral nerve cord ganglia are located in the anteriormost part of each segment in Z. yong and in some segments the ganglia seem to be placed in the intersegmental region (Fig. 8C, E). Except for the very elongated ganglia situated between segments 2 and 3, the ganglia of Z. yong are conspicuously round and compact. The elongated ganglia between segments 2 and 3 are almost as long as a segment, with nuclei scattered along the inter-ganglionic region and placed so close to each other that seem to form a single ganglion (Fig. 8C). In addition, less distinct aggregations of somata can be found in a middorsal position in each segment, especially in those segments with a middorsal spine (Fig. 8F).
Additional differences from F. kalenesos and C. styx include the transverse neurite bundles in the trunk (tn) of Z. yong which are only present in segments 2-9. They emerge from left and right sides of the ventral nerve cord at the level of the ganglia and encircle each segment. The transverse neurite bundles from segment 2 emerge from the ventral nerve cord in the anteriormost part of segment 3 and extend diagonally towards the posteriormost part of segment 2 (Fig. 8B, C, H). In segment 3 the transverse neurites are instead located in the posteriormost part of the segment. In segments 4-9 the transverse neurites are situated in the anteriormost part of each segment (Fig. 8B, E). The ventral nerve cord bifurcates in segment 10 into four neurite bundles (vncn) (Fig. 8B). Two of these neurite bundles extend laterally and encircle segment 10 while the other two extend towards segment 11 and form a ventral loop (Fig. 8B).
As in F. kalenesos and C. styx, Z. yong shows intrinsic α-tub-LIR in all the trunk spines. In addition, Z. yong has cuspidate spines (cu), which also show α -tub-LIR. Each cuspidate spine shows several neurites (cun) that extend radially from the point where the spine starts narrowing down towards the base of the spine and connect to the transverse neurite of the corresponding segment (Fig. 8, H, I). As in C. styx, Z. yong has a midterminal spine that shows α-tub-LIR (Fig. 8A).
Serotonin-like immunoreactivity
Serotonin-like immunoreactivity was studied in six specimens of Z. yong. 5HT-LIR is present in the brain neuropil, associated somata, ventral nerve cord and ventrolateral nerves (Fig. 9). Within the neuropil there are at least five immunoreactive rings where only two are incomplete ventrally (inr) (Fig. 9A). The first ring extends from a pair of 5HT-immunoreactive somata in ventromedial position (vms) (Fig. 9A, B). Five additional 5HT-LIR somata (asb) are present in the anterior brain region and project neurites into the neuropil (Fig. 9A, B, D). The ventral nerve cord shows at least five 5HT-LIR neurites that extend from segments 1-8, from which only two neurites extend posteriorly towards segment 11, forming a loop (Fig. 9A, B, E). Only a couple of 5HT-LIR somata were found associated with the ventral nerve cord (vncs), one on segment 5 and another one on segment 4, although not consistently present in all the studied specimens (Fig. 9A, B, E). Additionally, a pair of 5HT-LIR somata is present in a lateroventral position in segment 9 (Fig. 9A, B, E). Weak 5HT-LIR correlated with the transverse neurites of segment 9 was detected.
FMRFamide-like immunoreactivity
FMRF-LIR was studied in five specimens of Z. yong showing a consistent pattern in the brain neuropil and ventral nerve cord (Fig. 10A). Six pairs of FMRF-LIR somata (asb) are connected with the brain neuropil in the anterior region, and two pairs of somata (psb) in the posterior brain region (Fig. 10A–D). Some of the anterior somata are bipolar neurons with one neurite extended towards the introvert and another towards the neuropil (Fig. 10C, D). FMRF-LIR was also detected as a ring at the base of the mouth cone constituting a part of the mouth cone nerve ring (mcnr). The ventral nerve cord shows FMRF-LIR along its length, paired associated FMRF-LIR somata are present in segments 2, 3, 5 (two pairs), 6, 7, 9 and 10 (Fig. 10A, E–H). The position of the FMRF-LIR somata of the ventral nerve cord seem to be correlated with the position of the ganglia, except in segment 10 (Fig. 10A, F, H).